DESCRIPTION: (from the applicant's abstract) Many diseases are caused by mutations that lead to defective gene products or by disturbance in the regulation of gene expression that results in inappropriate synthesis. Human cells contain many structure- or function-related genes that can compensate the function of the mutant gene product. Unfortunately, most of these genes are either developmentally regulated, expressed in specific tissues, or regulated in a pathway by an upstream component whose function may be lost due to mutations in the gene. The ultimate goal of this project is to induce the expression of these function-related genes and apply it to gene therapy of human diseases using a novel technology, peptide nucleic acids (PNAs)-induced gene expression. PNAs are synthetic oligonucleotides with modified backbones. PNAs can bind to double-stranded structures at the PNA binding site. This D-loop structure has been found to have the capability to initiate gene transcription. In the PI's previous studies, binding of PNAs to a target site in the gamma-globin gene was found to induce both the GFP reporter gene and endogenous gamma-globin gene expression. However, the molecular mechanisms of PNA binding-induced gene expression have not been studied. The parameters that affect PNA-mediated in vivo gene expression also have not been investigated. In this proposal the principal investigator plans to study the molecular mechanisms of PNA binding-induced gene expression. The D-loop length requirement for gene transcription initiation will be defined. The transcription components involved in PNA binding-induced gene expression will be determined. The structure of the RNA transcripts from the PNA promoter will be studied. The correlation between PNA binding-mediated gene expression and DNA repair/replication will be investigated. The parameters affecting in vivo PNA gene induction such as PNA delivery efficiency and intracellular PNA binding also will be studied. The knowledge obtained from these studies will contribute to understanding the molecular basis of PNA binding-induced gene expression. The knowledge obtained from these studies will also be used to explore the possibility of using PNA technology to induce the expression of two human genes for treatment of two of the most common human diseases: induction of gamma-globin gene expression for sickle cell disease and induction of p21 gene expression for cancer. The knowledge obtained from this project will also help understand the molecular mechanisms of PNA binding-induced gene expression and provide direct evidence for treatment of many human diseases including genetic diseases and cancer. The aims are: To determine the molecular mechanisms of PNA-induced gene expression. To investigate parameters that affect PNA delivery efficiency and intracellular PNA binding. To explore the possibility of PNA binding-induced endogenous human gene expression.